Dodecyltrimethylammonium bromide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-12 to 2018-04-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for this test system (Sofuni, 1993)

Version / remarks:

1993

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dodecyltrimethylammonium bromide

EC Number:

214-290-3

EC Name:

Dodecyltrimethylammonium bromide

Cas Number:

1119-94-4

Molecular formula:

C15H34N.Br

IUPAC Name:

dodecyltrimethylazanium bromide

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Phenobarbital/ β-Naphthoflavone-pretreated rats

Test concentrations with justification for top dose:

1st Series (+/- S9): 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2)
2nd Series (+/- S9): 15.8, 50, 158, 500 and 889 µg/plate (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2)
3rd Series (-S9): 5, 15.8, 50, 88.9 and 158 µg/plate (S. typhimurium TA 98, TA 100, TA 1535)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultra-pure water
- Justification for choice of solvent/vehicle: The selection of the solvent for this assay was based on the available information from the preliminary solubility test. Ultrapure water showed best performance and was thus used for this experiment at a maximum concentration of 100 μL/plate.

Controls

Controls 1

Untreated negative controls:

no

Remarks:

The corresponding solvent control was used as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin (DAUN) in H2O

Remarks:

without S9 mix (TA 98)

Controls 2

Untreated negative controls:

no

Remarks:

The corresponding solvent control was used as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix (WP2 uvrA)

Controls 3

Untreated negative controls:

no

Remarks:

The corresponding solvent control was used as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix (TA 100, TA 1535)

Controls 4

Untreated negative controls:

no

Remarks:

The corresponding solvent control was used as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix (TA 1537)

Controls 5

Untreated negative controls:

no

Remarks:

The corresponding solvent control was used as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-AA) in DMSO

Remarks:

with S9 mix (TA 98, TA 100, TA 1535, TA 1537, WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
The incubation of plates was performed at 36 - 38 °C for 2 days.

NUMBER OF REPLICATIONS:
3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.

DETERMINATION OF CYTOTOXICITY
- Method: Reduced background lawn

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response (OECD TG 471, 1997).
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was considered, for example consistency of response within and between concentrations and (where applicable) between experiments.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 158 µg/ plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 500 µg/ plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test material on the agar plates accurred.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
DAUN (TA 98, -S9): 68 - 779 (mean: 243)
2-AA (TA 98, + S9): 112 - 3015 (mean: 738)
NaN3 (TA 100, - S9): 821 - 2376 (mean: 1550)
2-AA (TA 100, + S9): 437 - 3429 (mean: 1386)
9-AA (TA 1537, -S9): 247 - 1485 (mean: 736)
2-AA (TA 1537, +S9): 72 - 705 (mean: 293)
NaN3 (TA 1535, -S9): 351 - 2149 (mean: 867)
2-AA (TA 1535, +S9): 43 - 758 (mean: 168)
NQO (WP2 uvrA, -S9): 317 - 2275 (mean: 1677)
2-AA (WP2 uvrA, +S9): 154 - 696 (mean: 353)


- Negative (solvent/vehicle) historical control data:
Solvent (TA 98, -S9): 19 - 52 (mean: 36)
Solvent (TA 98, +S9): 20 - 58 (mean: 42)
Solvent (TA 100, -S9): 94 - 159 (mean: 118)
Solvent (TA 100, +S9): 90 - 172 (mean: 123)
Solvent (TA 1537, -S9): 4 - 11 (mean: 8)
Solvent (TA 1537, +S9): 6 - 16 (mean: 10)
Solvent (TA 1535, -S9): 15 - 38 (mean: 24)
Solvent (TA 1535, +S9): 6 - 28 (mean: 20)
Solvent (WP2 uvrA, -S9): 19 - 44 (mean: 30)
Solvent (WP2 uvrA, +S9): 28 - 47 (mean: 37)

Any other information on results incl. tables

In this study, three experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively. In the 3rd series no S9 mix was used. The results are summarised in the tables 1 - 3 below.

Table 1: Summary 1st Series

Test material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
		TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation (-S9)
H2O		30 ± 8	107 ± 6	19 ± 3	7 ± 2	30 ± 6
Test item	5.00	34 ± 10	107 ± 13	21 ± 3	7 ± 1	31 ± 7
	15.8	29 ± 9	117 ± 13	19 ± 5	7 ± 2	29 ± 6
	50.0	24 ± 5	110 ± 10	19 ± 5	9 ± 5	36 ± 5
	158	16 ± 3	36 ± 6^T	9 ± 1	7 ± 8	23 ± 2
	500	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T	21 ± 5^T
	1580	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T
	5000	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T
DAUN	2.00	317 ± 26
NaN3	2.00		1650 ± 38	764 ± 64
9-AA	50.0				1722 ± 293
NQO	2.00					1956 ± 145
With Activation (+S9)
H2O		40 ± 6	121 ± 9	22 ± 5	6 ± 3	47 ± 11
Test item	5.00	37 ± 9	112 ± 11	16 ± 4	6 ± 3	42 ± 6
	15.8	38 ± 6	121 ± 11	12 ± 4	7 ± 4	39 ± 3
	50.0	32 ± 9	137 ± 7	18 ± 2	10 ± 5	40 ± 10
	158	37 ± 7	100 ± 6	15 ± 4	11 ± 8	37 ± 16
	500	15 ± 3^T	0 ± 0^T	7 ± 1^T	7 ± 5^T	29 ± 3
	1580	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T
	5000	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T	0 ± 0^T
2-AA	2.00	932 ± 24	2225 ± 252
2-AA	5.00			82 ± 17	320 ± 51
2-AA	10.0					522 ± 141

Table 2: Summary 2nd Series

Test material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
		TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation (-S9)
H2O		31 ± 15	144 ± 8	26 ± 7	12 ± 4	35 ± 7
Test item	15.8	34 ± 4	142 ± 8	25 ± 11	10 ± 3	42 ± 7
	50.0	38 ± 7	135 ± 8	28 ± 5	13 ± 5	38 ± 10
	158	11 ± 3	62 ± 6	8 ± 0	8 ± 4	41 ± 6
	500	^T	^T	^T	^T	24 ± 3^T
	889	^T	^T	^T	^T	^T
DAUN	2.00	366± 25
NaN3	2.00		1640 ± 55	885 ± 22
9-AA	50.0				1947 ± 315
NQO	2.00					2495 ± 78
With Activation (+S9)
H2O		36 ± 10	126 ± 13	17 ± 5	11 ± 3	40 ± 8
Test item	15.8	32 ± 5	137 ± 13	10 ± 3	11 ± 3	34 ± 2
	50.0	41 ± 8	144 ± 5	17 ± 2	17 ± 2	44 ± 4
	158	39 ± 11	128 ± 8	10 ± 1	16 ± 4	39 ± 12
	500	^T	^T	6 ± 1	8 ± 3	34 ± 5
	889	^T	^T	^T	^T	22 ± 10^T
2-AA	2.00	351 ± 19	585 ± 17
2-AA	5.00			260 ± 11	132 ± 6
2-AA	10.0					215 ± 3

Table 3: Summary 3rd Series

Test material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
		TA 98	TA 100	TA 1535
Without Activation (-S9)
H2O		31 ± 12	104 ± 10	16 ± 4
Test item	5.00	36 ± 8	106 ± 13	20 ± 2
	15.8	36 ± 10	117 ± 1	20 ± 3
	50.0	33 ± 6	96 ± 8	18 ± 2
	88.9	26± 5	61± 7	19 ±5
	158	14± 2	26± 3^T	8± 2
DAUN	2.00	500± 44
NaN3	2.00		1450 ± 60	811 ± 72

NaN3: Sodium azide

2-AA: 2-Aminoanthracene

9-AA: 9-Aminoacridine

DAUN: Daunomycin

NQO: 4-Nitroquinoline-N-oxide

^T: Toxicity = red- bact. background lawn

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions dodecyltrimethylammonium bromide did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Executive summary:

Dodecyltrimethylammonium bromide was examined for its mutagenic activity according to OECD Guideline 471 in three series of an in vitro bacterial reverse mutation test employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA as indicator organisms.
The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/β-Naphthoflavone-pretreated rats was used. In this study, three experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used. Treatments of all tester strains were performed using dodecyltrimethylammonium bromide formulations prepared in ultra-pure water in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 μg/plate, plus vehicle and positive controls. The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values. The strain-specific positive controls, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Thus, the study is considered valid.
Following treatment with dodecyltrimethylammonium bromide, no precipitation of the test material on the agar plates occurred. Toxicity to the bacteria was observed at concentrations >= 158 μg/plate. Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to dodecyltrimethylammonium bromide in the absence and presence of S9 mix, thus the test item was not mutagenic under the described experimental conditions.